UDP-N-acetylglucosamine: .alpha.-6-D-mannoside .beta.1,6-N-acetylglucosaminyltransferase V (EC 2.4.1.155) is the Golgi enzyme responsible for the synthesis of the .beta.(1,6) branch structure of tri- and tetraantennary N-linked oligosaccharides. For brevity, this enzyme is abbreviated GlcNAc T-V herein. GlcNAc T-V activity has been found in many tissues and cell types.
Altered glycosylation of membrane glycoproteins and glycolipids is observed in mammalian cells transformed with diverse tumor viruses, carcinogens, or transfection with certain oncogenes. In some cases, there is a quantitative increase in a particular substituent, e.g., sialylation. In other instances, there is the reappearance of an oligosaccharide structure in the tumor which is normally only found in fetal tissue; for instance, certain Lewis histo-blood group antigens have been detected in adenocarcinomas.
Qualitative differences in oligosaccharides may also be observed in certain transformed cells. BHK fibroblasts transformed with polyoma virus or with Rous sarcoma virus display more highly branched complex N-linked oligosaccharides than do the corresponding normal cells. The expression of the .beta.1,6 branch structure (-[GlcNAc-.beta.(1,6)Man-.alpha.(1,6)Man]-) found in tri- and tetraantennary N-linked oligosaccharides is increased in the transformed cells. This has been correlated with a 2 to 3-fold increase in the specific activity of GlcNAc T-V. Transformation of murine cells with polyoma viruses, adenovirus, tumorigenic DNA and either the ras or the fps/fes oncogenes also resulted in increased GlcNAc T-V activity. By contrast, several other glycosyl transferases involved in N-linked glycosylation are unchanged in the transformed cells. The mechanism for the increased specific activity of GlcNAc T-V in transformed cells is not known.
The increase in the .beta.(1,6) branching of the cell surface-bound oligosaccharides has been associated, at least in some cases, with capacity for metastasis. Increased levels of .beta.-1,6 branching over the level in normal tissue has been observed for some human breast tumor tissues.
Certain mammalian glycosyltransferases from the N-linked glycosylation pathway have been purified and characterized. The enzymatic machinery for the glycosylation of proteins in mammalian cells is generally located in the membranes of the Golgi apparatus. .alpha.(1,3) mannoside .beta.(1,2) UDP-N-acetylglucosaminyltransferase I (GlcNAc T-I) (EC 2.4.1 101) and UDP-N-acetylglucosaminyltransferase II (GlcNAc T-II) (EC 2.4.1.143) have been purified from rabbit liver and rat liver, respectively. GlcNAc T-I has been purified 7000-fold from a Triton X-100 extract of rabbit liver acetone powder by two rounds of affinity chromatography over UDP-hexanolamine agarose, in the first round by elution with NaCl, and in the second round by elution with UDP (Oppenheimer and Hill (1981) J. Biol. Chem. 256:799-804). GlcNAc T-II (UDP-N-acetylglucosaminyl: .alpha.-D-mannoside .beta.(1,2) N-acetylglucosaminyltransferase II) was purified 60,000-fold from rat liver by Triton X-100 extraction of rat liver membranes, followed by chromatography over carboxymethyl-cellulose, hydroxylapatite, and sequential elutions using NaCl, UDP-GlcNAc and EDTA from 5-mercuri-UDP-GlcNAc-thiopropyl-SEPHAROSE, Affi-Gel (Bio-Rad Laboratories, Richmond, Calif.) blue affinity chromatography and finally UDP-GlcNAc-SEPHAROSE (Bendiak and Schachter (1987) J. Biol. Chem. 262:5775-5783).
The cDNA encoding a rat liver Golgi sialyltransferase (.beta.-galactoside .alpha.(2,6)-sialyltransferase (EC 2.4.99.1) has been cloned and sequenced (Weinstein et al. (1987) J. Biol. Chem. 262:17735-17743). The corresponding enzyme has been purified 23,000-fold from Triton CF-54 extracts of rat liver membranes by three rounds of affinity chromatography over CDP-hexanolamineagarose (Weinstein et al. (1982) J. Biol. Chem. 257:13835-13844). Soluble recombinant glycosyltranferases are described in U.S. Pat. No. 5,032,519, issued Jul. 16, 1991, incorporated by reference herein.
A portion of the work related to this invention has been published (Shoreibah et al. (1992) J. Biol. Chem. 267:2920-2927; Shoreibah et al. (1993) J. Biol. Chem. 268:15381-15385).